Desensitization of Signal Transduction

Continuous agonist stimulation of receptors usually leads to a decrease in receptor-mediated AC activity and cAMP levels. This process is termed desensitization. Desensitization, or a decreased response to the same or repetitive stimuli, is an important physiologic process and a well-described mechanism of receptor-signaling modulation that occurs in a variety of cell systems. There are two types of desensitization: homologous and heterologous. Homologous desensitization is agonist specific, whereas heterologous desensitization occurs when stimulation of another receptor or postreceptor stimulator leads to desensitization of the receptor. Defective desensitization could impair various steps in the signal transduction pathway, including receptor-G protein coupling, G protein activation by GTP, and G protein-effector interaction.159 Receptor mutations could lead to enhanced or reduced desensitization.160161 The reduction in cAMP generation observed during desensitization could be due to either a less efficient coupling of receptor to Gs or an enhanced efficiency of coupling of receptor to G,.159,162

Three independent mechanisms are responsible for desensitization: phosphorylation, sequestration, and down-regulation. The first step in desensitization involves an agonist-induced phosphorylation of the receptor, resulting in the functional uncoupling of the receptors from the effectors downstream in the signaling system. Thus, receptor phosphorylation is a primary mechanism leading to desensitization162"165 involving at least two kinases: phosphorylation by cAMP-dependent PKA and phosphorylation by P-adrenergic receptor kinase.166"168 PKA-mediated phosphorylation is cAMP dependent and plays a major role only in heterologous desensitization.163 p-Adrenergic receptor kinase-mediated phosphorylation is cAMP independent and is responsible for homologous desensitization.169 Phosphorylation may promote binding of other proteins to the receptor. These proteins are termed arrestins because they arrest signal transduction, perhaps by blocking G protein-receptor coupling.159167 After exposure to an agonist, receptors are sequestered in the plasma membrane within a few minutes. Sequestration refers to rapid, agonist-induced translocation of p-adrenergic receptor away from the plasma membrane to a distinct compartment deficient in Gs. Downregulation is a process biochemically distinct from sequestration and results in degradation of the receptor. Heterologous desensitization in lymphoma cells requires an increase in intracellular cAMP level.170

Prior exposure of human normal thyroid tissue to TSH either in vivo or in vitro causes desensitization of AC.171"174 TSH-induced homologous desensitization has been described in human and animal normal thyroid cells.172"180 After the cloning of the human TSH-R,181"184 the stable expression of TSH-R in nonthyroidal cells such as CHO cells has been used in desensitization studies.185 Although one study suggested that TSH stimulation failed to cause desensitization in CHO cells transfected with human TSH-R,185 a subsequent study did document that human TSH-R-transfected CHO cells do desensitize.186 The latter investigation is important because it documents that no specific thyroid factors other than increased levels of cAMP are required.186 Primary differentiated thyroid cancers, in general, desensitize similar to normal thyroid cells, whereas some metastatic tumors do not (Fig. 29-5). Loss of the desensitization ability could be responsible for the growth of thyroid cancers in TSH-sup-pressed patients and perhaps for metastatic disease. Desensitization of TSH-Rs in neoplastic cells is caused by increased levels of cAMP.186187 TPA itself had no direct effect on cAMP levels in all thyroid cancer lines, but it did cause desensitization of the AC response to subsequent stimulation by TSH.187 The activation of PKC appears to be responsible for the heterologous desensitization because staurosporine, a potent PKC inhibitor, abolished or inhibited the effect of TPA on desensitization.187 Prolonged TPA treatment leads to PKC downregulation as a result of depletion

cAMP (pmol/well)

FIGURE 29-5. Homologous desensitization of cyclic adenosine monophosphate (cAMP) to thyroid-stimulating hormone (TSH) stimulation in neoplastic thyroid cells from six patients. Cells were cultured in 24 well plates for 3 days and for the last 24 hours in medium lacking TSH and fetal calf serum until 95% confluence. These cells were preincubated for 4 hours in either control medium without TSH (CON) or in medium supplemented with TSH (10 mU/mL) and washed three times with phosphate-buffered saline, pH 7.4. The cells were then incubated for 30 minutes in control medium and in medium supplemented with TSH (10 mU/mL) and 1 mM isobutylmethylxanthine. Intracellular cAMP was then measured by radioimmunoassay. Each bar represents the mean ± SD of 18 experiments. Neoplastic thyroid cells (white bar) showed desensitization after pretreatment with TSH for 4 hours, whereas two of three metastatic cell lines (type I, hatched bar) had an increased cAMP level in response to TSH stimulation, and both failed to desensitize. The third metastatic cell line both failed to increase cAMP level in response to TSH and failed to desensitize (type II). P < .002. (Data courtesy of Tezelman S, Shaver JK, Grossman RF, et al. Mechanism of homologous and heterologous desensitization in human neoplastic thyroid cells. Unpublished personal data, 1996.)

of phorbol ester-binding studies in many cell types.188 The effects of PKC downregulation on TSH-stimulated iodide organification have been demonstrated in porcine thyroid cells.189 Like TPA, EGF had no direct effect on cAMP levels, but it did cause desensitization of cAMP production to subsequent TSH stimulation.190 The EGF-induced desensitization of TSH-AC signal transduction system was abolished by coincubation with EGF-R antibody (EGF-R monoclonal antibody 528) and by genistein.190 Desensitization apparatus, phosphodiesterase expression, and CREB may be activated in functioning thyroid adenomas.191193

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